小分子封闭提高固定化青霉素酰化酶的稳定性

为提高青霉素酰化酶的催化性能和热稳定性,在酶组装过程中添加小分子试剂对介孔泡沫硅载体表面过量的活化位点进行封闭。详细考察了小分子添加质量分数和种类对青霉素酰化酶负载率、催化活力及热稳定性的影响。实验结果得到:经精氨酸封闭的固定化酶活力提高至1.92倍;甘氨酸封闭的固定化酶5 h的50℃热稳定性提高至2.9倍,甘氨酸和谷氨酸封闭的固定化酶50℃热处理25 h仍保持87.9%和82.2%的残余活力;甘氨酸和谷氨酸封闭的固定化酶最适催化pH值向中性偏移且对pH值的耐受性增强。结果表明,在青霉素酰化酶共价组装过程中添加合适的小分子封闭能显著提高酶的催化性能和热稳定性。     

Ag/P(St-MMA)纳米复合高分子微球固定化青霉素酰化酶的研究

通过溶剂热法和无皂乳液聚合相结合,制备了P(St-MMA)高分子纳米微球.并以吸附沉积的方式在其表面沉积了Ag金属纳米粒子,最后将青霉素酰化酶共价连接在微球表面.初步研究了微球直径、银的质量分数等因素对固定化酶活力的影响.结果显示随着微球直径减小,固定化酶的偶联率和活力逐渐增加;银纳米粒子最多将固定化酶的偶联率和活力分别提高了42%和72%,固定化酶的最大表观活力(以干重记)达到了1 869 u/g,明显高于其它高分子载体固定化青霉素酰化酶的活力;实验证明银纳米粒子在青霉素水解过程中没有催化活力,但能大大提高青霉素酰化酶的催化活力.     

介孔纳米孔道中单维交联酶策略固定青霉素酰化酶

为解决酶固定化过程中稳定性较差的问题,将单维交联酶策略引入青霉素酰化酶的固定化中,通过在介孔泡沫硅载体(MCFs)表面初步吸附作用将酶固定,再用对本醌对酶分子进行交联,从而制得单维交联酶聚体。实验结果显示:单维交联酶聚体(CLEA)的稳定性有明显提高,最适温度从游离酶的45℃上升到55℃,催化温度的适用范围更宽,在50℃下热处理25 h仍保持74.4%活力,在测试操作稳定性方面,连续操作15次,其酶活保持在61.9%。     

以壳聚糖为载体固定化青霉素酰化酶的研究

介绍了以壳聚糖为载体固定化青霉素酰化酶需首先制备壳聚糖颗粒 ,使用戊二醛、甲醛、乙二醛 3种活化剂处理所得的壳聚糖颗粒 ,确定了以戊二醛为活化剂交联其上的氨基共价结合青霉素酰化酶的固定化方法。从戊二醛的浓度、pH值、固定化时间、固定化pH值、酶用量等条件摸索了最佳固定化条件 ,获得了酶活力为4 0 0 0 0U/ (g·h)、回收率为 5 0 %左右的固定化青霉素酰化酶。     

磁性微粒对青霉素G酰化酶的固定化研究

将无机磁性粒子Fe3O4与有机材料海藻酸钠结合起来制成一种复合的磁性微粒,并将其进行表面修饰,通过化学共价法来固定青霉素G酰化酶。通过扫描电镜等对微粒进行形态学观察,并用傅立叶红外图谱表征微粒表面修饰基团。酶学性质研究表明,该微粒固定化的青霉素G酰化酶的最适pH值为7.5,最适温度为40℃。固定化酶与底物的亲和力有所降低,但是稳定性显著提高。重复催化研究结果表明,固定化酶具有比游离酶更广泛的温度及pH值适用范围,并且具有良好的热稳定性、可循环使用性和贮存稳定性。     

Acinetobacter johnsonii G2细胞的固定化及其非水相介质中催化合成葛根素糖苷

利用海藻酸钠包埋法固定化Acinetobacter johnsonii G2细胞,在非水相介质中生物催化葛根素合成葛根素糖苷,考察了细胞的固定化条件、转化条件以及固定化细胞的操作稳定性。结果表明,最佳固定化条件为:海藻酸钠质量分数为2%,氯化钙质量分数为2%,细胞包埋量的体积分数为50%;最佳转化条件为:温度为40℃,p H为6.47,二甲基亚砜体积分数为20%。在最佳条件下,葛根素在催化体系中质量浓度提高至31.92 g/L,产率达93%,且固定化细胞的重复使用稳定性好。与游离细胞相比,固定化细胞对p H、温度及有机溶剂表现出更强的稳定性,且在非水相介质中催化效率更高。     

氨基树脂固定化S-腺苷甲硫氨酸合成酶的研究

以氨基树脂为载体对S-腺苷甲硫氨酸(SAM)合成酶进行固定化,优化了酶的固定化条件并对固定化酶的性质进行了研究。优化的固定化条件为:戊二醛体积分数5%、SAM合成酶添加量20mg·g-1、固定化时间5h。所制备的固定化SAM合成酶的酶活力为476.8U·g-1,酶活力回收率为74.5%。与游离SAM合成酶相比,固定化SAM合成酶的稳定性大幅提高,在50℃孵育5h酶活力仍保留61.2%,而游离SAM合成酶则完全失活;在pH值为6.0~6.5、8.0~9.5的缓冲溶液中,固定化SAM合成酶也更加稳定;固定化SAM合成酶连续催化反应10批次,酶活力保留86.3%;固定化SAM合成酶在4℃储存30d,酶活力保留81.4%。固定化SAM合成酶米氏常数KATPm=0.14mmol·L-1,KLm-Met=0.28mmol·L-1。     

磁性聚合物微球固定化青霉素G酰化酶用于催化合成头孢克洛

利用反相悬浮技术制备出平均孔径和比表面积分别为13.0 nm和123.6 m~2/g的磁性聚合物微球,其具有超顺磁性,饱和磁化强度为6.04 emu/g。磁性微球固定化青霉素G酰化酶在乙二醇-磷酸盐缓冲溶液共溶剂体系中催化7-氨基-3-氯-3-头孢烯-4-酸(7-ACCA)与D-苯甘氨酸甲酯(D-PGM)合成头孢克洛,20℃反应2 h时,头孢克洛产率为33.0%,合成与水解比(S/H)为0.13;而在相同反应条件下使用游离青霉素G酰化酶,产率和合成与水解比分别为17.0%和0.08。同时考察了酶用量、反应温度以及溶剂对磁性固定化酶催化合成头孢克洛性能的影响规律。     

纤维素酶催化与三液相萃取偶联制备盾叶薯蓣皂苷元

利用三液相萃取与酶催化转化耦合技术对盾叶薯蓣中薯蓣皂苷进行水解,并分离提取薯蓣皂苷元。考察了几种甾体皂苷和水解下的葡萄糖在三液相中的分配、酶的分配以及酶活力在三液相体系中的保留情况,并对比了三液相、有机相、水相中纤维素酶催化转化与薯蓣皂苷元的生成情况。实验结果表明,正己烷/1,4-二氧六环/硫酸铵三液相体系能够满足酶催化和产物分离同步进行的要求,使薯蓣皂苷元分配在正己烷相,底物和酶保留在1,4-二氧六环相,大部分葡萄糖进入硫酸铵相,反应进行96h薯蓣皂苷元的得率为69.4%,比有机相、水相酶催化分别提高一倍和27.6倍,显示出耦合技术的明显优势。     

双交联剂体系氨基酰化酶交联聚体的制备

采用酶交联聚集体技术（CLEAs）制备氨基酰化酶(EC 3.51.14)交联聚集体,优化了制备条件。90%（体积比）乙醇为沉淀剂,戊二醛和乙二醇二缩水甘油醚（EGDE）作为混合交联剂,以酶、戊二醛和EGDE的质量比为1.00∶0.75∶2.00,在30 ℃下交联12 h。制备所得氨基酰化酶CLEA酶活保留率为63.42%。氨基酰化酶经固定化后热稳定性得到增强,热失活过程由自由酶的一阶指数失活模型变为二阶指数失活模型,分子间亚基的结合力得到显著加强。自由酶在37 ℃下保存24 h后几乎损失了所有活力,而氨基酰化酶CLEA则保留了43%的酶活力,在57 ℃下保存24 h后仍有32%的酶活力保留。此外,氨基酰化酶CLEA重复使用性能得到明显提升,在重复水解底物10次后仍保留了72%的酶活力。     

Improvement of the catalytic performance of immobilized penicillin acylase through assembly of macromolecular reagents in nanopore to create a crowding environment

Macromolecular reagents were co-assembled with penicillin acylase (PA) and immobilized in mesocellular siliceous foams (MCFs) to resemble living cells. Types and concentrations of macromolecules were studied. The catalytic characteristic and stability of PA preparations were also investigated. PA assembled with dextran 10 k in MCFs showed maximum specific activity, 1.32-fold of that of the solely immobilized PA. The optimum pH of dextran and BSA derivatives shifted to neutrality, and the optimum temperature increased by 10 °C. Also, Km of BSA derivative of PA declined 56.7% compared to solely immobilized PA, while the Kcat/Km of PA assembled with BSA was enhanced to 147%. After incubation at 50 °C for 6 h, residual activity of PA assembled with BSA exhibited 53.0%. The ficoll derivative showed 82.8% of its initial activity at 4 °C after 8-week storage. The results indicated that macromolecular reagents assembled with PA in MCFs could dramatically improve the catalytic performance and stability of immobilized enzyme.     

海藻酸钠-明胶协同固定化S-腺苷甲硫氨酸合成酶的研究

以海藻酸钠和明胶为载体,对S-腺苷甲硫氨酸合成酶进行固定化。再用戊二醛对其进一步交联,增强固定化酶的稳定性。考察了海藻酸钠和明胶质量分数、CaCl2质量分数、酶和载体比例以及交联剂戊二醛体积分数等因素对固定化酶的影响。结果表明,最佳固定化条件为:海藻酸钠质量分数2.0%、明胶质量分数1.0%、CaCl2质量分数4.0%、固定化酶量为2.5 g/L凝胶、戊二醛体积分数0.6%。交联固定化酶热稳定性得到大幅度提高,在50℃下保温5 h仍保留72%的活力,而游离酶则完全失活。交联固定化酶在碱性溶液中的稳定性较高,在pH=8.0～9.0的缓冲液中4℃保温10 h酶活性仍保留87%以上。将交联固定化酶用于S-腺苷甲硫氨酸的合成,连续反应8批次后酶活性仍保留65%。     

Covalent immobilization of penicillin G acylase onto amine-functionalized PVC membranes for 6-APA production from penicillin hydrolysis process. II. Enzyme immobilization and characterization

This article describes the covalent immobilization of penicillin G acylase (PGA) onto glutaraldehyde-activated NH₂-PVC membranes. The immobilized enzyme was used for 6-aminopenicillanic acid production from penicillin hydrolysis. Parameters affecting the immobilization process, which affecting the catalytic activity of the immobilized enzyme, such as enzyme concentration, immobilization's time and temperature were investigated. Enzyme concentration and immobilization's time were found of determine effect. Higher activity was obtained through performing enzyme immobilization at room temperature. Both optimum temperature (35°C) and pH (8.0) of immobilized enzyme have not been altered upon immobilization. However, immobilized enzyme acquires stability against changes in the substrate's pH and temperature values especially in the higher temperature region and lower pH region. The residual relative activities after incubation at 60°C were more than 75% compared to 45% for free enzyme and above 50% compared to 20% for free enzyme after incubation at pH 4.5. The apparent kinetic parameters K_M and V_M were determined. K_M of the immobilized PGA (125.8 mM) was higher than that of the free enzyme (5.4 mM), indicating a lower substrate affinity of the immobilized PGA. Operational stability for immobilized PGA was monitored over 21 repeated cycles. The catalytic membranes were retained up to 40% of its initial activity after 10.5 working h. © 2012 Wiley Periodicals, Inc. J Appl Polym Sci, 2012     

基于大分子拥挤原理的介孔二氧化硅中青霉素酰化酶的共价组装

To improve the covalent immobilization of penicillin acylase （PA）, macromolecular crowding theory was applied to its immobilization. Influence of mass ratio of enzyme to the silica, as well as, activation time with glutaraldehyde on the activity of assembled PA, was studied. In the mesopores, the effect of fl-cyclodextrin （β-CD） on the immobilization of the enzyme was also investigated. It was remarkable that the coupled yield and relative activity reached 99.5% and 92.3%, respectively, when penicillin acylase assembled covalently in the mesopores. The results here indicate that mimicked macromolecule crowding could significantly ameliorate the performance of covalently immobilized PA.     

Immobilization of penicillin acylase on copolymer of butyl acrylate and ethylene glycol dimethacrylate

The effects of glutaraldehyde, enzyme concentrations and reactants volumes, ionic strength, pH value and carrier particle diameter on immobilization of penicillin acylase onto acrylic carriers were studied. The activity of immobilized enzyme preparations was also studied over a range of pH values and temperatures and thermal and pH stabilities were determined. The use of the immobilized preparation for penicillin G hydrolysis in a batch reactor was investigated. The immobilized enzyme gave a significant reduction in hydrolysis time compared to hydrolysis by the native enzyme.     

转谷氨酰胺酶在聚丙烯微孔膜上的固定化

研究了转谷氨酰胺酶在聚丙烯微孔膜上的化学固定化的影响因素,确定了最佳固定化工艺条件,即为:第一步光照反应6 min,单体质量分数为20%,第二步光照时间25 min,接枝率最高可达35.2%;己二胺质量分数为25%,胺烷基化时间150 min,胺烷基化温度60℃;戊二醛质量分数3%,戊二醛作用时间45 min;酶液浓度10 mg/mL,固定化时间20 h,固定化温度4℃,固定化酶膜的活力最高可达游离酶的45%.并研究了温度、pH、金属离子对固定化酶膜的酶学性质的影响,其贮存性能和操作稳定性也做了初步研究.     

Covalent crowding strategy for trypsin confined in accessible mesopores with enhanced catalytic property and stability

Chemically modified macromolecules were assembled with adsorptive trypsin in mesoporous silica foams (MCFs) to establish covalent linkage. Effects of catalytic properties and stability of immobilized trypsin were examined. The addition of chemically modified protein (BSA) and polysaccharide (ficoll) to the immobilized trypsin exhibited high coupled yield (above 90%) and relative activities (174.5% and 175.9%, respectively), showing no protein leaching after incubating for 10 h in buffers. They showed broader pH and temperature profiles, while the half life of thermal stability of BSA-modified preparation at 50 °C increased to 1.3 and 2.3 times of unmodified and free trypsin, respectively. The modified trypsin in aqueous-organic solvents exhibited 100% activity after 6 h at 50 °C. The kinetic parameters of trypsin preparations and suitable pore diameter of MCFs warranted compatibility of covalent modification for substrate transmission. The covalent crowding modification for immobilized trypsin in nanopores establishes suitable and accessible microenvironment and renders possibility of biological application.     

海藻酸钠固定化S-腺苷甲硫氨酸合成酶的制备及其性质研究

研究了以海藻酸钠包埋法制备固定化S-腺苷甲硫氨酸（sAM）合成酶的条件,并考察了固定化酶的酶学性质。结果表明,最适固定化条件为：海藻酸钠质量分数3％、CaCl2质量分数4％、SAM合成酶加量（以每克海藻酸钠计）75mg、固定化时问30min,在此条件下,固定化酶活力回收率达到42％。固定化酶热稳定性较好,在50℃下保温5h仍保留69％的酶活力,而游离酶则完全失活;固定化酶在碱性条件下的稳定性较好,在pH值7．5～9．0的缓冲溶液中4℃下保温10h仍保留84％以上的酶活力;将固定化酶用于SAM的合成,连续反应5批次后,仍保留82％的酶活力。